Sensors for detecting pulmonarydiseases from exhaled breath

Dina Hashoul and Hossam Haick

Affiliation: Dept of Chemical Engineering, Russell Berrie Nanotechnology Institute, and the TechnionIntegrated Cancer Center, Haifa, Israel.

Correspondence: Hossam Haick, Technion Israel Institute of Technology, Haifa, 32000003, Israel. E-mail:hhossam@technion.ac.il

@ERSpublicationsDetection of volatile organic compounds from exhaled breath by nanomaterial-based sensors is a newdiagnostics frontier in the screening of pulmonary diseases. http://bit.ly/2JoBKXn

Cite this article as: Hashoul D, Haick H. Sensors for detecting pulmonary diseases from exhaled breath.Eur Respir Rev 2019; 28: 190011 [https://doi.org/10.1183/16000617.0011-2019].

ABSTRACT This review presents and discusses a new frontier for fast, risk-free and potentiallyinexpensive diagnostics of respiratory diseases by detecting volatile organic compounds (VOCs) present inexhaled breath. One part of the review is a didactic presentation of the overlaying concept and thechemistry of exhaled breath. The other part discusses diverse sensors that have been developed and usedfor the detection of respiratory diseases (e.g. chronic obstructive pulmonary disease, asthma, lung cancer,pulmonary arterial hypertension, tuberculosis, cystic fibrosis, obstructive sleep apnoea syndrome andpneumoconiosis) by analysis of VOCs in exhaled breath. The strengths and pitfalls are discussed andcriticised, particularly in the perspective in disseminating information regarding these advances. Ideasregarding the improvement of sensors, sensor arrays, sensing devices and the further planning of workfloware also discussed.

IntroductionRespiratory diseases are often diagnosed in later stages, reducing the chance of effective treatment [1].Diagnosis currently includes physical examination followed by a series of tests that include chemical,imaging, endoscopic, immunological and genomic procedures among others [2, 3]. Extensive screeningand detection of respiratory diseases at an early stage can dramatically decrease morbidity and mortality [4],because this enables prompt intervention/treatment, with the prospect of achieving the best possibletherapeutic outcome for the patient. Moreover, these programmes may enable diagnosis of high-riskconditions for the development of a particular disease [1]. By identifying and managing these high-riskconditions, it may be possible to act preventatively, thereby reducing the incidence of disease occurrence [5].Monitoring of individuals identified as being high-risk cases is important in terms of determining thepoint at which the disease begins to progress, notably in subjects where transformation from a benign to amalignant state occurs (as in lung cancer), and planning interventions for such individuals [6]. Theaccuracy and/or accessibility of tests available in currently existing programmes fails to reach the desiredlevels, whereas new developments (e.g. sputum tests, radiography and computed tomography (CT) scans)are costly [7]. In other instances, the tests can be more invasive (endoscopy, pulmonary catheterisation,biopsy and bone marrow tests) and, therefore, run the risk of complications to the patients screened and/or require special facilities (such as CT) with healthcare professionals operating the instruments [8]. Theideal test for respiratory diseases should have high-accuracy, low cost, be noninvasive, easily repeatable at

Copyright ©ERS 2019. This article is open access and distributed under the terms of the Creative Commons AttributionNon-Commercial Licence 4.0.

This article has supplementary material available from err.ersjournals.com

Provenance: Submitted article, peer reviewed.

Received: Feb 11 2019 | Accepted after revision: May 13 2019

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specific intervals and nontechnical. A distinct advantage is for the procedure(s) to have minimal impacton the daily activities of the person being screened [9].

Volatile organic compounds as biomarkers for respiratory diseasesA new frontier for fast, risk-free and potentially inexpensive diagnosis of respiratory diseases is based onvolatile organic compounds (VOCs), i.e. organic compounds that have high vapor pressure at ambientconditions. The rationale rests on the fact that VOCs show distinct and immediate changes whenpathological conditions arise, altering the body’s biochemistry by one or a combination of the followingprocesses: oxidative stress, cytochrome p450, liver enzymes, carbohydrate metabolism and lipidmetabolism [2, 10]. Part of these VOCs, which appear both in normal and abnormal cells, are mixtures ofdistinctly different compositions [11]. The remainder come from exclusively abnormal cells. Theparticularly significant feature that can be exploited in this approach is that each disease has its ownunique VOC pattern, and therefore the presence of one disease would not screen out others [12]. TheseVOCs can therefore be detected: 1) directly from the headspace (i.e. the mixture of VOCs trapped abovethe abnormal cells in a sealed vessel); or 2) in exhaled breath, blood or other body fluids, somethinghighly dependent on their tissue–blood and blood–air partition coefficients [13]. Therefore, isolation anddetection of VOCs in these body fluids can serve as a pathway for the early detection of respiratory andother diseases. The monitoring of respiratory diseases by breath analysis is noninvasive and breathsampling can be carried out without the need for specialist settings and specialist technical expertise.

Several spectrometry and spectroscopy techniques have been used to collect, detect and analyse exhaledVOCs of respiratory diseases [14–18]. Frequently used techniques include proton transfer reaction-mass

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FIGURE 1 a) Overview of the working principal of nanomaterial-based sensors array. b) Differentnanomaterial-based sensors. 1) Chemiresistor based on monolayer-capped nanoparticles; 2) chemiresistorsbased on single-wall carbon nanotubes; 3) chemiresistor based on conducting polymers; 4) chemiresistorbased on metal oxide film; 5) quartz microbalance with selective coating; 6) colorimetric sensor; and7) surface acoustic wave sensor. Reproduced from [11] with permission from the publisher.

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spectrometry (MS), selected ion flow tube (SIFT)-MS, ion mobility spectrometry, laser spectroscopy andgas chromatography (GC), which is considered the most utilised method [11]. In GC techniques, theexhaled breath is collected and usually stored in inert bags or sorption tubes. After desorption, VOCs areassessed and analysed by GC which is usually followed by MS or flame ionisation detection [19]. VOCsare separated based on their chemical properties being consecutively ionised and separated by theirmass-to-charge (m/z) ratio [2]. While GC-MS-based techniques are powerful in detecting disease-relatedVOCs, they unfortunately require expensive equipment, high levels of expertise to operate the instruments,considerable time and effort for sampling and analysis, and a need for preconcentration techniques [10].

These approaches have been used to identify the VOCs distinctive of several respiratory diseases, includingchronic obstructive pulmonary disease (COPD), asthma, lung cancer, pulmonary arterial hypertension(PAH), obstructive sleep apnoea syndrome (OSAS), tuberculosis (TB), cystic fibrosis (CF) andpneumoconiosis. An updated list compiling the characteristic VOCs for these respiratory diseases is shownin table S1.

Sensor arrays for disease detection in exhaled breathTo overcome the challenges associated with spectroscopic and/or MS techniques for breath analysis ofrespiratory diseases, chemical sensors have been adopted. Detection of the disease-related VOCs fromexhaled breath can be achieved using two main chemical sensing strategies. The first is based on a selectivemechanism in which a chemical sensor is designed to interact and detect the presence of a singlecompound in exhaled breath [2]. This approach, though quite sensitive, is cumbersome due to thecomplex synthesis of separate and highly selective nanomaterials for the detection of each VOC, mainlywith nonpolar targets. Moreover, there are currently no individual unique VOCs that are specific to anyparticular disease [20]. The second approach involves an array of broadly cross-reactive sensors along withmethods of pattern recognition (figure 1a) [11, 21]. In contrast to the selective sensing method, thesensors array approach, bioinspired by the sense of smell, is capable of detecting a compendium of VOCs.Each sensor in this approach responds to a range of VOCs which allows the sensing and analysis ofindividual components from a mixture of compounds [22]. The underlying mechanism of this approachdepends on the nature of the sensors (figure 1b); for example, chemiresistors change their electricalresistivity due to sorption of VOCs on the organic film, or by steric changes within the sensing layeraffecting the charge transfer from/to the inorganic nanomaterial (figure 1b) [20]. Acoustic sensors detectchanges in the propagation (velocity and amplitude) of acoustic waves through or on the surface of thesensor’s coating material due to sorption of VOCs [23]. Colorimetric sensors are based on indicators,specifically chemoresponsive dyes, which chemically react and change colour on exposure to VOCs,thereby identifying the exposed species [24].

The most widely used approach for sensors array is possibly based on conductive polymers, which operateon electrical resistance changes from steady state induced by the attachment of VOCs to the sensor [25].While polymer-based chemiresistors offer several advantages (e.g. low power consumption, small size, lowoperating temperature and low cost), their sensitivity depends on the type of coating; they also show adrift in baseline due to polymer instability [25, 26]. Nevertheless, selection of a sensors array type dependson the physical characteristics that are optimal for clinical purposes; sensor arrays with low recovery timeare not optimal for screening purposes [25]. Table 1 lists representative diseases and related types of

TABLE 1 Sensor arrays in the diagnosis of respiratory diseases

Disease Sensor type References

Lung cancer CBPC, MO, SWNTs, SiNW FET, MCMNPs, QMB,colourimetric

[27, 28, 29, 30, 31, 32, 33, 34,35, 36]

COPD CBPC, QMB, MO [37, 38, 39, 40, 41, 42]Asthma CBPC, MO [37, 43, 44, 45, 46, 47, 48]PAH MCMNPs, colourimetric [49]OSAS CBPC [50, 51, 52]CF CBPC [53, 54, 55]TB MO, SWNTs, MCMNPs, [56, 57, 58, 59, 60, 61]Pneumoconiosis CBPC [62]

COPD: chronic obstructive pulmonary disease; PAH: pulmonary arterial hypertension; OSAS: obstructivesleep apnoea syndrome; CF: cystic fibrosis; TB: tuberculosis. CBPC: carbon black polymer composite; MO:metal oxide; SWNTs: single-walled carbon nantotubes; SiNW FET: silicon nanowire field effect transistors;MCMNPs: monolayer-capped metal-coated nanoparticles; QMB: quartz microbalance.

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sensor arrays used for their detection. In the following sections, this table will be extended to discuss theuse of sensor arrays conjugated with pattern recognition methods as diagnostic tools for differentrespiratory diseases.

Chronic obstructive pulmonary diseaseCOPD is characterised by oxidative stress and production of VOCs secreted by the lungs [38]. Diagnosis isbased on identifying characteristic symptoms and lung functioning parameters [23, 63]. Current diagnostictools poorly reflect the severity and other distinctive features of the disease [63].

Nuclear magnetic resonance and GC-MS analysis of exhaled breath condensates (aerosolised nonvolatileparticles contained in the fluid lining of the airway) and noncondensated exhaled breath concentrations oflactate, acetate, propionate, serine, proline and tyrosine are raised, but valine and lysine are lower thannon-COPD controls [37, 45, 64–66]. Relying on these breath-print signatures, other research groups havedeveloped sensors to detect COPD-related VOCs in exhaled breath. In a cross-sectional study of 100patients with asthma and COPD, breath VOCs were analysed by a sensors array based on 32 derivatives ofpolymer/carbon black sensors [37]. Principle component analysis (PCA) of the sensing signals had 88%accuracy for distinguishing fixed asthma from COPD, and 83% for classic asthma, and the detectionaccuracy was not confounded by smoking status [37]. Since both COPD and asthma patients have chronicairway inflammation, the results might include overlapping features, in the sense that COPD patientscould be misdiagnosed as asthmatics and vice versa. Hence, it is essential to clearly discriminate COPDfrom asthma, especially in elderly people who have a higher probability of adverse reactions to differentclasses of inhaled agents or systemic corticosteroid [67, 68]. Thus, a combined system consisting of anarray of quartz crystal microbalance (QMB) sensors coated with six derivatives of metal-basedmetalloporphyrins linked with a GC system found nine VOCs that were significantly correlated withCOPD, of which two positively correlated with COPD and seven negatively correlated with COPD [38].These results showed that the following were significantly increased in healthy subjects: limonene;butylated hydroxytoluene (BHT); 2-propanol; benzene, 1,3,5-tri-tert-butyl-; hexane, 3-ethyl-4-methyl-;hexyl ethylphosphono-fluoridate; and 1-pentene, 2,4,4-trimethyl. The alkanes decane and 6-ethyl-2-methyldecane were raised in COPD patients [38]. Parallel to the GC-MS analysis, the cross-validated modelprovided the correct classification of 26 out of 27 COPD patients, and five out of seven control subjects,with an accuracy of 91% [38].

AsthmaSymptoms of atopic asthma often begin in early childhood and mostly improve, or even disappear, atpuberty, but can relapse later in life [68]. Analysis of exhaled breath may, therefore, be used to assessinflammation and oxidative stress in the respiratory tract, thereby providing a diagnostic approach to thecondition [69, 70]. The most common breath analysis approach is based on the detection and monitoringof exhaled nitric oxide fraction (FeNO) [70]. Indeed, increased exhalation of nitric oxide (NO) as a result ofinterleukin (IL)-13-induced induction of NO synthase in the airway epithelium have been widelydocumented in asthmatic patients. As a result, asthmatic patients exhale >30 ppbv of NO, whereas ahealthy population exhales lower concentrations [71].

Portable NO-selective sensors are already routinely used to detect asthma, generally being sensitive to NOlevels of <1 ppb, with a relatively rapid response time [45]. Nevertheless, raised NO concentrations havebeen reported in other diseases, including hypertension, arthritis, lung diseases, bronchiectasis and CF, inaddition to inflammatory bowel disease of the colon and small intestine [72]. Consequently, a patternrecognition approach of exhaled breath is more likely to be fruitful for predicting asthma than a singlebiomarker. Indeed, sensors array have given a higher degree of diagnostic accuracy for asthma thanexhaled NO or lung function [14, 44, 73]. For example, a cross-sectional study with polymer/carbon blacksensors array could distinguish between 30 patients with COPD, 20 patients with mild-to-severe asthma,20 healthy smokers and 20 healthy nonsmokers [45]. The breath-prints of patients with COPD andasthmatics have been compared, and the data analysed by PCA and canonical linear discriminant analysis.Moreover, cross-validation with the leave-one-out method has been used to estimate the accuracy, showingthat the polymer-based sensors could successfully discriminate patients with mild-to-severe asthma fromthose with COPD, healthy smokers and healthy nonsmokers with accuracies of 96%, 93% and 95%,respectively [45]. Similarly, polymer/carbon black sensors array successfully separated mild asthma fromyoung controls; however, it failed to distinguish severe from mild asthmatics [66]. PAREDI et al. [14] haveshown that the VOC profile can assess asthma severity and control. In addition to the collective analysis ofbreath-prints, raised ethane levels have been recorded in the breath of steroid-naïve asthmatics comparedto subjects treated with steroids. Moreover, ethane was found to be higher in patients with severe asthmacompared with patients with mild asthma, suggesting that ethane might be a preselected marker for thedetection of asthma.

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Lung cancerLung cancer is typically asymptomatic in its early stages and, as such, most of the cases are diagnosed inlater stages when treatment is no longer effective [74]. The 5-year survival rate increases dramatically from10% to 80% if the disease is detected at the early stages [75].

Numerous GC-MS and proton transfer reaction-MS studies have examined the profile of VOCs in lungcancer; >1000 trace VOCs have been found in the exhaled breath of lung cancer patients at concentrationsranging from parts per million by volume to parts per trillion by volume [6, 9, 15, 76–81]. Typicalexamples are isoprene, methanol, acetone and 2-propanol (appearing in all human breath samples),acetonitrile, furan, 2-methyl furan (primarily found in smokers) and many others [82]. Hence, efforts havebeen invested in analysing exhaled breath as a simple noninvasive method of the early detection of lungcancer [83, 84]. Relying on these findings, DI NATALE et al. [27] reported 100% classification of patientswith lung cancer versus healthy subjects by using eight QMB sensors coated with differentmetalloporphyrins. Similarly, the same sensor array was used to detect lung cancer in a pool of 36 healthycontrols, 28 patients with lung cancer and 28 patients with diverse lung diseases [34]. The sensorsresponse was analysed using PCA and discriminate analysis, combined with partial least square ; it couldclassify between the groups with 85%, 92.8% and 89.3% sensitivity, respectively.

Surface acoustic wave (SAW) sensors were used as a detector for the breath analysis of 42 volunteers,including 15 healthy subjects, 20 patients with lung cancer and seven patients with chronic bronchitis.This array was a SAW coated with a film of isobutylene regarded as the detector, with the other sensorbeing used as a reference. After calibration of the sensors, breath samples were collected in Tedlar bagsand absorbed on solid-phase microextraction fibres for preconcentration. The VOCs extracted from thethermal desorber column were absorbed on the SAW sensors. Frequency response (Hz) and thecorresponding retention times were recorded. Using artificial neural network analysis, the sensors couldcorrectly diagnose patients with lung cancer from exhaled breath with 80% sensitivity and specificity [28].

Despite their sensitivity and good response time, SAW sensors are temperature sensitive, such that certainanalyte compounds are affected by the different sensor coatings [10]. Therefore, others have tried to verifythe potential of these different types of nanoarray sensors to detect lung cancer. For example, acolorimetric sensors array was designed for noninvasive lung cancer detection of exhaled breath of 49patients with nonsmall cell lung cancer, 21 healthy volunteers and 73 patients with different pulmonarydiseases, including COPD [29]. Each colorimetric sensors array was composed of 36 chemically sensitivespots with different sensitivities to volatile compounds. The data gave 73.3% sensitivity and 72.4%specificity for the diagnosis of lung cancer. In a following study, breath samples were taken from 229volunteers divided into a control group of individuals at high risk of developing lung cancer, subjects withintermediate lung nodules, and untreated patients with lung cancer validated by biopsy. All groups wereexamined by a colorimetric sensors array and compared with breath signatures of eight binary groups forthe identification and characterisation of lung cancer with high sensitivities and specificities. The resultingmodel could discriminate between different lung cancer histologies with 90% sensitivity; however,prediction and differentiation between healthy volunteers and patients with lung cancer was less accurate(70%) [35]. Arguably, the disposable features of the colorimetric sensors might be a limitation inreal-world applications.

With these challenges in mind, chemiresistors based on monolayer-coated metal nanoparticles have beenused to detect and monitor lung cancer as having advantages over other sensing techniques, such as: alarger surface-to-volume ratio of the sensors, operation at room temperature, lower detection limits for theVOCs of interest (sub-ppb), lower operating voltage, a wider dynamic range, faster response and recoverytimes, higher tolerance to humidity and compatibility with standard microelectronic industry [2, 30].Using this approach, the group of HAICK and co-workers. [31–33, 36] successfully discriminated early andlate stages of lung cancer with 88% accuracy. Moreover, this sensors array could distinguish between smallcell lung carcinoma and nonsmall cell carcinoma, as well as in differentiating between subhistologies ofadenocarcinoma and squamous cell carcinoma with 93% and 88% accuracy, respectively. Furthermore, in astudy that included 144 breath samples from 39 patients with advanced lung cancer, one gold nanoparticle(GNP) sensor could differentiate between patients with lung cancer after surgery, as well as monitoring theresponse of patients to therapy with an accuracy of 59%. Discriminant factor analysis of the collectiveresponses from the nanoarray could monitor changes in tumour response during therapy and also indicatelack of any further response to therapy with a success rate of 85% [85]. Using the same sensors array,these authors managed to detect lung cancer at early onset and monitor breath volatolomics after lungcancer resection. Moreover, patients with lung cancer and volunteers with benign nodules before and aftersurgery could clearly be differentiated by DFA maps [32]. Ultimately, the nanomaterial-based sensorsarray distinguished between pre-surgery and post-surgery lung cancer states yielding an 80% classificationaccuracy [32]. In another study by the same group, exhaled breath was analysed in the diagnosis of

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epidermal growth factor receptor (EGFR) mutation in patients with lung cancer [36]. Ananomaterial-based sensors array composed of 40 cross-reactive, chemically diverse chemiresistors basedon organically stabilised spherical GNPs, and single-walled carbon nanotubes, were used to discriminatepatients with lung cancer who harboured the EGFR mutation from those with wild-type with an accuracyof 83%. Nanoarray sensors also showed that patients with early lung cancer could be discriminated frompatients with benign pulmonary nodules, with a sensitivity, specificity and accuracy of 75%, 93% and 87%,respectively (figure 2).

Pulmonary arterial hypertensionPAH is a progressive cardiopulmonary disease characterised by the extensive occlusion of small tomid-sized pulmonary arterioles, as well as structural alterations in the vascular wall that eventually lead toright heart failure [12, 13, 86]. Although a wide range of therapeutic agents have been established for themanagement of PAH, it remains incurable, with lung transplantation continuing to be the main treatmentin severe cases [87]. Most diagnostic methods involve assessment of the cardiac structure or estimation ofpulmonary artery pressure; however, PAH does not manifest until pulmonary vascular disease is advanced [86].Accordingly, screening for PAH in a high-risk population would aid early diagnosis and intervention,thereby improving patient outcomes [88, 89].

In PAH, changes in the exhaled volatolome could result from the pathophysiological process ofremodelling in arterioles, as well as the compensatory mechanisms accompanying the development ofPAH [13]. Moreover, the physiological changes in the lungs and heart, and fluctuations in the pulmonarycirculation, could be other sources of volatolomic alterations [90]. Consequently, many groups are tryingto use changes in the exhaled breath pattern to detect PAH in its earlier stages. Exhaled breath of PAHpatients has been examined by SIFT-MS in predetermined training and validation sets [90]. A total of 31PAH patients and 34 controls were enrolled in the study. The breath of patients with PAH had raisedconcentrations of 2-nonene, 2-propanol, acetaldehyde, ammonia, ethanol and pentane compared withcontrol subjects, whereas 1-decene and 1-octene were significantly lower. The model also gave 86.1%accuracy in the training phase and 79.3% in the independent validation set. In contrast, there was noassociation between levels of ammonia in the breath and plasma (plasma levels of ammonia were similarin both groups). Ammonia is generated by the breakdown of nucleic acids, polyamines and amino acids,mainly glutamine. Moreover, PAH patients showed higher glutaminolysis, thereby generating moreammonia in the exhaled breath seen in this study, although not enough to affect whole bloodconcentration [90].

While SIFT-MS offers real-time VOC detection and quantification of exhaled breath, its clinicalapplications suffer from high cost, with high levels of expertise being required to operate the instrument,and a long time needed to complete the analysis of breath samples. Thus, efforts continue to be made todevelop nanoscale sensors for the rapid detection of VOCs in exhaled breath. In fact, COHEN-KAMINSKY

et al. [49] have established a proof of concept that GNP-based sensors can successfully detect and classifyPAH cases in a pool of 22 patients and 23 healthy controls with an accuracy of 92%. Similarly, acolorimetric sensors array was used as a diagnostic method for the discrimination of patients with lung

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FIGURE 2 Discriminant factor analysis plots calculated from the responses of the nanoarray sensors for a) earlylung cancer and benign pulmonary nodules and b) patients with lung cancer with and without the epidermalgrowth factor receptor (EGFR) mutation. Each point represents one patient. The positions of the mean valuesare marked with an unfilled square; the boxes correspond to the first and third quartiles, and the error barscorrespond to the SD. CV1: first canonical variable. Reproduced from [36] with permission from the publisher.

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cancer from other common lung diseases, wherein 20 patients with PAH were enrolled as a control group [29].The results showed that the breath signatures of patients with lung cancer differed from PAH, idiopathicpulmonary fibrosis, COPD and healthy controls. In the validation set of the nanoarray, the sensors coulddiscriminate between the diseases with a sensitivity of 73.3% and a specificity of 72.4%. The results werenot influenced by sex, age, histology or smoking history.

TuberculosisDiagnosis of TB remains a major global public health challenge, with its social burden increasing becausemany patients may also be infected with HIV. The rates of multidrug-resistant TB are increasing [58, 91].In 2010, there was an estimated incident case count of 8.8 million active TB infections, resulting in1.5 million deaths [57]. Current diagnostic methods rely on either insensitive smear microscopy orsensitive, but lengthy, microbiologic culture, unlikely to be used in poorly resourced centres [56, 59]. Morerecently, the Xpert MTB/RIF assay, a fully automated sample-to-answer nucleic acid amplification test, hassignificantly improved sensitivity compared with smear microscopy [58]. However, this assay requires asputum sample or an invasive sample from patients that cannot expectorate. Its high cost also limits it usein poor and resource-limited countries where TB is rampant [61]. Hence, development of a rapid,affordable and noninvasive assay is needed for TB screening, especially in developing countries.

PHILLIPS et al. [60] analysed the breath of 226 symptomatic high-risk patients using GC-MS, pointing outseveral biomarkers of active pulmonary TB. They suggested biomarkers in oxidative stress products, suchas alkanes and alkane derivatives, and volatile metabolites of Mycobacterium tuberculosis, such ascyclohexane and benzene derivatives. Their results differentiated between positive and negative TB with85% overall accuracy, 84% sensitivity and 64.7% specificity, using C-statistic values [60]. In an attempt touse the distinctive breath-prints of TB patients in a point-of-care setting, a metal-oxide-based sensorsarray was tested in a pilot study for the detection of TB in Bangladesh on 30 participants and 194 in thevalidation set [57]. The response was analysed and validated using traditional sputum smear microscopyand culture on Lowenstein–Jensen media. The results showed a sensitivity of 93.5% and a specificity of98.5% in the validation set. Moreover, the sensors array had a sensitivity of 93.5% and a specificity of85.3% in discriminating healthy controls from patients with TB, and a sensitivity of 76.5% and specificityof 87.2% in identifying patients with TB within the entire test population [57].

NAKHLEH et al. [56] used arrays of molecularly modified GNP and molecularly modified single-walledcarbon nanotubes for the detection of active TB. Their study included 64 individuals in whomM. tuberculosis was proven by culture. The control group consisted of two main subcategories, 67 healthyvolunteers and a group of 67 subjects in whom TB was suspected but had had negative smears, cultureand GeneXpert MTB/RIF. The two groups were age- and sex-matched, but differed in their smokinghabits, HIV infection rates and treatment for TB [56]. The classification ability of the sensors wasevaluated using receiver operating characteristic (ROC)-derived Youden’s index as a cut-off point (bestsensitivity+specificity−1) as a binary classifier threshold for the training set. Three single-walled carbonnanotubes sensors modified with a layer of polyaromatic hydrocarbon derivative showed >80% accuracy inthe training set, whereas the other nine showed either <80% accuracy or a random classification.Alternatively, the chemiresistor based on dodecanethiol-capped GNPs correctly classified 121 out of the138 in the training set, with an accuracy of 88%, a sensitivity of 85% and a specificity of 89%. In thevalidation set, the same sensor scored a sensitivity of 90%, a specificity of 93% and an accuracy of 92%.Moreover, the sensor’s function was unaffected by possible confounding factors, including smoking habits,HIV infection and antibiotic treatment (figure 3) [56].

Another group used a sensors array composed of eight metalloporphyrin-coated QMB sensors to assessthe exhaled breath of patients with TB during treatment. Breath samples of 51 patients with TB and 20controls were analysed before and 2, 7, 14 and 30 days after therapy. The sensors response was validatedand correlated with clinical and microbiological measurements on sputum samples [58]. The sensorsscored 93% accuracy in distinguishing TB cases from controls; additionally, serial measurements of VOCsalso showed signal changes during TB treatment among patients.

In addition to TB, nanomaterial sensor arrays have been successfully employed for the detection of otherrespiratory infections including sinusitis, ventilator-associated pneumonia and invasive pulmonaryaspergillosis in prolonged chemotherapy-induced neutropenia [92–98].

PneumoconiosisApproximately 15% of lung diseases are attributed to pneumoconiosis, a lung condition that is associatedwith occupational exposure leading to inhalation of dust, silica, asbestos or smoke [62]. The high-riskpopulation includes shipyard workers, construction workers, asbestos textile workers and asbestos miners [23].Although asbestos has been gradually banned in many countries, mortality due to asbestos exposure

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continues to increase in many developed countries. However, diagnosis of early-stage pneumoconiosisremains difficult in clinical practice [19, 23]. The inhaled dust particles are phagocytosed by alveolarmacrophages, resulting in reactive oxygen species being produced. Consequently, lipid peroxidation occursand hence screening for lipid peroxidation-related VOCs should be useful in diagnosing pneumoconiosis [62].

YANG et al. [62] analysed the exhaled breath of 34 subjects with pneumoconiosis and 64 healthy patientsusing a polymer/carbon black sensors array. The prediction model based on linear discriminant analysisindicated that the discriminations of the sample yielded a specificity of 88.0%, a sensitivity of 67.9% andan accuracy of 80.8% in the training set. In the test set, sensitivity was 66.7%, specificity was 71.4%, andaccuracy was 70.0% by linear discriminant analysis, suggesting that nanoarray sensors based on polymer/carbon are potential valuable in screening for pneumoconiosis.

Obstructive sleep apnoea syndromeOSAS is a common disease associated with an increased risk for cardiovascular disorders [50]. Despite theintroduction of several screening tools, the diagnosis of OSAS still needs to be confirmed bypolysomnography, and hence the use of expensive instruments by trained personnel is required limiting itslarge-scale application [51].

In an attempt to utilise sensors array technology for the detection of OSAS, the polymer composite-basedsensors were utilised in the discrimination of OSAS between healthy controls showing adequate sensitivityand specificity (93% and 70%, respectively) [50]. In another study, the same sensors array was employedto test the breath-prints of 18 children with OSAS and 10 non-OSAS subjects with habitual snoring (aged6–11 years). The exhaled biomarker pattern of patients with OSAS was discriminated from that of controlsubjects (p=0.03, cross-validation accuracy: 64%), ROC curve analysis (area: 0.83) showed 78% sensitivityand 70% specificity [99]. In a more recent study, 136 subjects (20 obese non-OSAS, 20 hypoxic OSAS, 20nonhypoxic OSAS, and 20 nonhypoxic COPD versus 56 healthy controls) were tested using seven quartzcrystals. Their collective breath samples were analysed, and controls were distinguished from othersscoring 100% correct classification. Moreover, the sensors array identified 60% of hypoxic, and 35% ofnonhypoxic patients with OSAS [100]. Similarly, a 100% correct classification was obtained when controland COPD groups were compared. Finally, INCALZI et al. [52] used the same quartz-based sensors array toshow that breath-prints of patients with OSAS significantly change after a single night of continuouspositive airways pressure and it largely depends upon studied comorbidities like diabetes mellitus,metabolic syndrome and chronic heart failure.

Cystic fibrosisCF is characterised by inflammation and oxidative stress; thus, monitoring of airway inflammation andoxidative stress can be helpful in the diagnosis and monitoring of CF, especially since inflammation arisesbefore clinical symptoms appear [101].

The currently available techniques for measuring inflammation and oxidative stress in the airways arebronchoscopy, bronchoalveolar lavage and biopsy; however, these techniques are too invasive for repeatedroutine use, especially in children [19]. The oxidative stress that accompanies inflammation in CF andother respiratory diseases leads to the formation of distinctive volatile substances in the breath, which hasled to increasing interest in exhaled breath analysis for the detection of CF. FeNO is the most extensively

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0.0

1.0

0.8

0.6

0.4

0.2

0.0

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1- Specificity 1- Specificity

0.8 1.0 0.0 0.2 0.4 0.6 0.8

Smoking AUC 56%

HIV status AUC 54%Treatment AUC 55%

1.0

AUC 0.948

FIGURE 3 a) Dot plots response of a single molecularly modified gold nanoparticle. Each symbol represents a single sample. The dashed linerepresents Youden’s cut-off point, and the dotted lines represent the cut-off points to rule in and rule out tuberculosis. Samples from thevalidation set with responses lower than the threshold were classified as tuberculosis positive (open stars) or nontuberculosis positive (closedstars) according to Youden’s cut-off point. b) Receiver operating characteristic curve of the sensor. c) Receiver operating characteristic curves ofthe sensor when comparing smoking, HIV and treatment status. AUC: area under curve. Reproduced from [56] with permission from the publisher.

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studied marker in exhaled breath, and has been proved to be helpful clinically in some pulmonarydiseases, including CF [6, 53]. Nevertheless, monitoring NO has several limitations, most noted in thatFeNO is largely a marker of allergic inflammation, thereby limiting its use in nonallergic patients. Next tomonitoring preselected unique markers, it is possible to assess the profiles of VOCs in exhaled air [101].Indeed, the exhaled breath of 64 patients with CF and 21 with primary ciliary dyskinesia were analysedusing the polymer composite-based sensors array along with PCA analysis [55]. A cross-validated ROCcurve was constructed; breath profiles of patients with CF showed a significant difference from controls(p=0.001) and from patients with primary ciliary dyskinesia (p=0.005). The sensors’ response could alsodifferentiate between CF and primary ciliary dyskinesia with or without a number of well-characterisedchronic pulmonary infections [55]. Furthermore, the sensors array had the ability to discriminate betweenpatients with CF suffering from chronic pulmonary Pseudomonas aeruginosa infection from those withouta chronic pulmonary infection. However, the results indicated that it was impossible to detect any overalldifference between chronically and nonchronically infected patients [55]. It was also impossible todifferentiate nonchronically infected patients with CF from patients with CF having other chronicpulmonary infections with other pathogens, such as Achromobacter xylosoxidans, Stenotrophomonasmaltophilia or a species of the Burkholderia cepacia complex. The authors explained these findingsregarding possible bacteria-specific VOCs that patients with CF with chronic pulmonary infection emit intheir exhaled breath [55].

MCGRATH et al. [53] found that patients with CF with an acute exacerbation had lower levels of exhaledisoprene compared with controls [53]. Moreover, when these patients were treated with antibiotics, theirisoprene levels increased to normal levels. Ethane levels were also raised in steroid-naïve patients with CFcompared with steroid-treated patients [53]. Overall, the data indicate that VOC profiling could be usefulin assessing and following up exacerbations, and for rapid detection of P. aeruginosa in patients with CF.

Future perspectives and concluding remarksSensor arrays are potentially becoming convenient devices for physicians in the detection and monitoringof therapy of patients with respiratory diseases. Improvement in sensor technologies, machine-learningmethods, disease-specific reference libraries and databases, in addition to the identification ofrespiratory disease biomarkers, have all contributed to the advance in diagnostic methods based onexhaled breath [6, 102]. Nevertheless, before VOC profiling can become a potent clinical tool, considerablymore work is needed to allow it to be applied in clinical practice. Several different stages have to beaddressed in every part of the development of chemical sensing systems for disease diagnostics. Theseinclude clinical and engineering aspects, as well as commercial and ethical issues, which could take years [2].An important step is for the extensive validation of the currently available VOC profiles [20]. This shouldbe done by worldwide population studies that can statistically confirm many of the suggested patterns.Another approach ought to rely on basic research studies that can connect breath profiles to biologicalpathways of a particular disease [30, 103, 104]. Other validation aspects to consider, for instance, are thesubstantial differences in methodology, such as breath collection and sampling techniques across differentstudies. A standardised methodology is required to take advantage of the different datasets [105]. Forexample, the relative humidity of exhaled breath may vary and influence measurements; water absorptionreduces the sensitivity of metal oxide sensors by preventing electron donation to the surface charge layer.Alternatively, gold or platinum metal monolayer-capped nanoparticle chemiresistors have low sensitivity towater [104]. Another example might be related to the breath manoeuvres; a recent study on chronicrhinosinusitis showed that patients that had underlying asthma presented some confounding influence [20].This could also relate to differences in sampling procedure due to asthmatic individuals having moredifficulties in breathing (e.g. change in exhalation rate, shallow breaths, etc.) [20].

Environmental influences and the effects of ambient and background VOCs also have to be taken intoaccount. For example, during the offline procedure of exhaled VOC collection, Tedlar bags could releaseVOCs into the collected breath, and storage in Tenax tubes might disturb the composition of the breathsample [106]. Concerning reproducibility, instrumental and classification repeatability between differentsensor models must also be considered to allow identical profile and output [107]. In regard to controlgroup selection, an appropriate group of individuals should be selected, taking into account sex, smokinghabits, fasting and comorbidities, among other confounding factors. Although age and sex are known tomodify individual VOCs, this does not seem to affect the overall profile as analysed by the sensors array [5].However, the presence of comorbidities, such as renal failure, heart diseases and several forms of cancer,along with smoking habits, hamper the composition of exhaled VOC and therefore the resultingbreath-print [108]. Such confounding factors may prevent the diagnosis of interest and should always betaken into consideration [5, 107, 109]. Lastly, one of the most crucial aspects of nanomaterial-based sensortechnology is data analysis; the digital outputs generated by the sensors have to be analysed and

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interpreted in order to provide useful information. The choice of method depends on the type of availableinput data acquired from the sensors and the type of information that is sought [110].

Sensor response to VOCs can be analysed by pattern recognition algorithms to classify different casesindividually, in which the principal component reduction and subsequent pattern recognition bydiscriminant analysis are the most frequently used types of raw-data analysis for their responses [5]. Othertechniques are also used for data analysis, such as machine-learning algorithms and neural networks [111].These techniques mimic the cognitive process of the human brain, containing interconnected dataprocessing algorithms that work in parallel [110]. The results of the artificial neural network data analysisare usually in the form of a percentage match of identification elements in a given breath sample withthose of VOC patterns seen in a training set-up. The diversity of analytical techniques that are availablemay hinder the standardisation of sensors array technologies, and consequently special care must be givento avoid overfitting the training data and validation sets.

Acknowledgements: The authors acknowledge Yoav Broza (Dept of Chemical Engineering, Technion, Haifa, Israel) forhis constructive criticism and proof-reading of this manuscript. D. Hashoul acknowledges the Neubauer DoctoralFellowship Fund for a PhD scholarship.

Conflict of interest: None declared.

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